Indices of lower airway inflammation in children monosensitized to house dust mite after nasal allergen challenge

Background: There are few available data assessing the united airway disease and its systemic aspects in children. With this study, we aimed to investigate the inflammation markers of upper and lower airways before and after nasal allergen challenge in mite sensitive children with different clinical expression of the allergic disease. Methods: Four study groups were formed: rhinitis only, without bronchial hyper‐responsiveness (R, n = 10), rhinitis with asthma (R + A, n = 22), atopic asymptomatics (AA, n = 8) and nonallergic healthy controls (C, n = 10). Blood eosinophils, nasal and sputum eosinophils, sputum eosinophil cationic protein (ECP) and cys‐LTs, and serum ECP levels were measured before and 24 h after nasal allergen challenge. Results: The groups were comparable in terms of age and gender. Cumulative symptom scores recorded during and 1 h after nasal challenge were not significantly different between patients with R, R + A and AA groups. At T₂₄, the children belonging to R, R + A and AA showed significant increases in nasal eosinophils (P < 0.01, P < 0.001, and P = 0.01, respectively), sputum eosinophils (P = 0.01, P < 0.001, and P < 0.05, respectively) and blood eosinophils (P < 0.01, P < 0.001, and P < 0.05, respectively). Similarly, increases in sputum ECP (P < 0.01, P < 0.001, and P = 0.07, respectively) and sputum cys‐LT levels (P = 0.07, P < 0.001, and P < 0.05, respectively) were detected in children belonging to these three groups at T₂₄. Sputum eosinophils significantly correlated with blood eosinophils (r = 0.54, P < 0.001) and sputum ECP (r = 0.58, P < 0.001) at T₂₄. Conclusions: This study showed that nasal allergen challenge increased markers of eosinophilic inflammation in both upper and lower airways of children monosensitized to mites, even before the onset of clinical symptoms.     

屋尘螨变应原重组体免疫治疗的实验研究

目的：建立屋尘螨哮喘动物模型，比较分别用屋尘螨粗浸液、重组Derp2（rDerp2）进行免疫治疗的疗效，为变态反应性疾病的免疫治疗提供新的思路。方法：近交系清洁级BALB／c小鼠，腹膜内注射（i．p．）屋尘螨变应原粗浸液与灿（OH）3生理盐水的混悬液致敏小鼠，建立哮喘模型；治疗组在致敏后分别给予i．p．屋尘螨粗浸液、rDerp2溶液进行免疫治疗后，鼻腔滴入（i．n．）屋尘螨粗浸液予以激发；阴性对照组从致敏，阳性对照组在致敏后，均同步生理盐水处理。用ELISA方法检测各组实验动物血清中IgE、IgG1、IgG2a的水平，以及支气管肺泡灌洗液（BALF）与脾细胞培养后上清液（SCCS）中细胞因子IL-4、IFN-γ水平，计数BALF中细胞总数与分类细胞数，并制备肺组织病理切片以观察其病理改变。结果：添加铝佐剂的屋尘螨变应原致敏的小鼠在特异性变应原的连续激发下，肺组织产生明显的炎症反应，表现为BALF中细胞总数增加以及炎症细胞所占比例及绝对计数升高，且以分泌Th2型细胞因子如IL4为主；血清IgE、IgG1水平升高；肺组织病理切片观察到支气管、细支气管上皮下和小血管周围有明显的炎性细胞浸润，以嗜酸性粒细胞为主，气道上皮结构紊乱，有部分脱落，上皮下充血水肿明显，肺泡间质增厚，也可见到炎性细胞浸润。给予相应免疫治疗后，肺组织炎症反应明显减轻，表现为BALF中细胞总数减少，炎症细胞数减少，且其和SCCS中n1型细胞因子如IFN-γ占主导；血清IsE、IsG1水平降低，IgG2a水平升高；肺组织病理切片观察到细胞浸润程度明显减轻，气道结果基本恢复正常。结论：本实验研究成功地建立了小鼠哮喘模型。分别用屋尘螨粗浸液及rDerp2进行免疫治疗均显示良好疗效，但前者比后者疗效好。     

PACAP38 improves airway epithelial barrier destruction induced by house dust mites allergen

PurposeThis study aimed to investigate the mechanism of PACAP38 on house dust mite (HDM)-induced asthmatic airway epithelial barrier destruction.MethodsThe HDM-induced asthma mice model and 16HBE cell model was established respectively. The enzyme linked immunosorbent assay (ELSIA), cell count and immunohistochemical assay were performed on mice in control group, HDM group and PACAP38 + HDM group.The cAMP/PKA activity, p-CREB and total CREB expression, TEER and the FITC-DX were investigated on cells in control-16HBE group, HDM-16HBE group and PACAP38 + HDM-16HBE group.ResultsThe levels of IL-4 and IL-5 in the HDM group were significantly higher than those in the control group (P < 0.05), while the above indexes in the PACAP38 + HDM group were lower than those in the HDM group (P < 0.05). E-cadherin, β-catenin, ZO-1 and occludin in the control group were highly immunoreactive in airway epithelial cells, whereas connexin staining was attenuated after HDM induction. The TEER level, cAMP levels and PKA activity were decreased, while FITC-DX transmittance was increased in HDM-16HBE group (P < 0.05) compared with the control-16HBE group.ConclusionPACAP38 could reduce the airway inflammation, weaken the AJC protein heterotopia and activate cAMP/PKA signaling pathway in HDM-induced asthma, which indicate that PACAP38 may be an important contributor in HDM-induced asthma.     

Effects of volatile constituents of a rosemary extract on allergic airway inflammation related to house dust mite allergen in mice

Perilla leaf extract is known to have anti-inflammatory properties. Recently, we have demonstrated that rosmarinic acid, a polyphenolic liquid component in perilla, inhibits the allergic airway inflammation induced by house dust mites (HDMs) in vivo. The purpose of this study was to evaluate the effects of intratracheal (i.t.) exposure to volatile constituents of a rosemary extract (VR), gaseous components in perilla, on a murine model of allergic asthma induced by HDM. C3H/HeN mice were treated 7 times weekly with i.t. exposure. The HDM allergen challenge elicited a pulmonary eosinophilic inflammation accompanied by an increase in the lung expression of interleukin (IL)-5, IL-13, and eotaxin. VR inhibited increases in the number of eosinophils, neutrophils, and mononuclear cells around the airways and those in the bronchoalveolar lavage fluid. VR exposure also significantly suppressed the expression of IL-13 enhanced by HDM allergen. These results suggest that i.t. exposure to VR can, at least partially, prevent allergic airway inflammation induced by HDM. The preventive effect is associated with inhibition of the enhanced local expression of IL-13.     

Murine allergic respiratory responses to the major house dust mite allergen Der p 1.

BACKGROUND: Although many studies have examined chronic asthma, limited data exist on acute immunopathogenic events induced by allergens. The aim of the study was to investigate the acute cellular, serologic and histopathologic events in airway inflammation produced by intranasal challenge of mice sensitised to the major house dust mite allergen Der p 1. METHODS: C57BL/6 mice were immunised subcutaneously with Der p 1 in alum. Mice were bled and challenged intranasally with Der p 1 on day 14 and killed on day 17. Lungs were fixed in situ, processed and stained with haematoxylin and eosin. The degree of inflammation and eosinophil infiltration was quantified by image analysis. Specific IgE was determined by passive cutaneous anaphylaxis. Cells from spleen and draining lymph nodes were cultured for 24 h with Der p 1, and IL-3/GM-CSF released into supernatants was measured by bioassay. RESULTS: Intranasal challenge of sensitised mice induced eosinophilic influx into the large and small airways and the alveolar regions of the lung, mucus plugging and in severe cases numerous Charcot-Leyden crystals. The quantitation of the inflammation induced by different sensitisation and challenge doses showed that optimal inflammation could be produced using only 1 microg of allergen for both sensitisation and challenge. The degree of inflammation was not related to the titre of IgE antibody and was indeed produced in its absence. T cell reactivity of spleen cells to the allergen was decreased suggesting cell migration or inactivation. CONCLUSIONS: Mice sensitised and challenged intranasally with as little as 1 microg of Der p 1 produced an extensive pulmonary eosinophilic inflammation which shared many of the features of the inflammation found in asthma. The small amount of allergens required and the use of intranasal challenge should provide a useful model.     

Distinct requirements for interleukin-6 in airway inflammation induced by diesel exhaust in mice

To clarify the possible role of interleukin-6 in aggravation of inflammatory responses in diesel exhaust-exposed mice, we compared the infiltration of inflammatory cells and the production of chemokines between interleukin-6-deficient and wild-type mice following 0, 1.0, or 3.0 mg diesel particles/m³ diesel exhaust inhalation for 4 weeks. Exposure to diesel exhaust significantly increased the number of inflammatory cells and the amount of CCL17 and CXCL3 in bronchoalveolar lavage fluids from wild-type mice, but not in interleukin-6-deficient mice. These findings suggest that interleukin-6 plays a critical role in airway inflammatory responses induced by diesel exhaust inhalation.     

Airway remodelling and inflammation in sheep lungs after chronic airway challenge with house dust mite

BACKGROUND: Remodelling of airway walls is a significant morbidity factor in patients suffering from chronic asthma. The relationship between airway remodelling and the inflammatory response is not well defined. Sheep have been used extensively to model airway disease in humans and represent a suitable model to examine airway remodelling. OBJECTIVE: The aim of the present study was to develop a model for airway remodelling in sheep after repeated challenge with a relevant human allergen to assess the relationship of airway remodelling with inflammation. METHODS: Repeated challenges of house dust mite (HDM) extract or saline (control) were administered to local lung segments of sheep for a period of 6 months. After the last challenge, lung tissues from both challenged and unchallenged lung compartments of the same sheep were compared using morphometric image analysis and (immuno) histological studies. RESULTS: All HDM-challenged sheep developed significant bronchoalveolar lavage eosinophilia during challenge. At the end of the challenge period, significant increases in airway collagen and airway smooth muscle content were found in a proportion (3/7) of the HDM-challenged sheep. Hyperplasia of goblet cells and epithelial cells were observed in small bronchi and bronchioles exposed to allergen. Irrespective of airway remodelling changes, all HDM-challenged, but no saline-challenged sheep, displayed significant increases in mast cells in alveolar septa and airway walls of challenged lungs compared with untreated lung compartments of the same sheep. Significant increases were also observed in CD5 and gamma delta T cell subpopulations in all allergen-exposed lung parenchyma. CONCLUSION: A proportion of atopic sheep develop typical airway remodelling changes after chronic allergen challenge, which is not directly related to the level of allergic inflammation.     

Distribution of house dust mite allergen: comparing house dust mite allergen levels in dust samples collected from different sites on living room floors with smooth coverings

BACKGROUND: The distribution of house dust mite allergen (Der p1) in living rooms with smooth floor coverings, as measured in the middle compared with the border of the floor was investigated. It was hypothesized that activity causes displacement of Der p1, from the middle towards the border. METHODS: Dust samples from the middle and border of 50 floors with smooth coverings were collected and analysed on Der p1 content in a standardized way. RESULTS: The Der p1 exposure expressed as per unit area (ng/m2) showed that border samples contained significantly more Der p1 compared with middle samples (median: 2.57 vs 0.27, respectively, P = 0.023). Presence of pets and presence of more than two inhabitants increased the difference. When expressed as per unit weight of dust (ng/g), significant differences were only detected when comparing Der p1 content of samples collected in households with three or more inhabitants [median: 2 (border) vs 53 (middle), respectively; P = 0.035]. CONCLUSIONS: The Der p1 is unequally distributed on living room floors with smooth coverings, most likely because of displacement of dust from the middle towards the border due to activity. Expression as ng/g of dust and ng/m2 could not obviously be interchangeable.     

Influence of mattress characteristics on house dust mite allergen concentration

BACKGROUND: Exposure to a high level of house dust mite allergens (HDMAs) is considered as a risk factor for HDM sensitization and development of asthma in genetically disposed people. Mattresses are one of the most important sources of HDMA in people's living environment. OBJECTIVE: The aim of this study was to evaluate the association between mattress characteristics and HDMA concentrations on mattresses. METHODS: Dust samples of mattress surfaces were taken to evaluate the level of Der p 1 allergen. All participants filled in a questionnaire about the type of mattress, the type of covering (upper layer) of the mattress, dwelling characteristics and cleaning habits. Humidity and temperature of the bedroom were measured at the time of dust sampling. RESULTS: One hundred and sixty-eight questionnaires were filled in. Synthetic upper layer of the mattress was associated with a higher level of Der p 1 compared with cotton upper layer (2.6 vs. 0.8 microg/g Der p 1). Moreover, higher relative humidity (RH) was associated with significant higher concentrations and density of Der p 1. CONCLUSIONS: Two factors were associated with lower levels of Der p 1 found on mattresses, namely: a cotton upper layer of the mattress compared with a layer of synthetic material and lower RH at the time of sampling. As far as we know, the association between type of upper layer and concentration of Der p 1 has not been described before and could lead to the formulation of practical advices in order to reduce HDMA concentrations on mattresses.     

Diesel exhaust particles exert acute effects on airway inflammation and function in murine allergen provocation models

BACKGROUND: Epidemiologic studies show that sudden surges in ambient particulate matter (PM) levels can trigger acute asthma exacerbations. Although diesel exhaust particles (DEPs) act as an adjuvant for allergic sensitization, this is a delayed response and does not explain acute PM effects on airway hyperreactivity (AHR). OBJECTIVE: Our aim was to determine the acute effects of DEPs on AHR using a mouse model. METHODS: Three protocols were developed, 2 of which require OVA sensitization, whereas the third was OVA independent. In the mild sensitization protocol BALB/c mice receive intraperitoneal OVA without alum and are then challenged with aerosolized OVA with or without DEPs. In the postchallenge model DEPs are delivered after OVA challenge to animals sensitized by intraperitoneal OVA plus alum. In the third protocol nebulizer DEPs were also delivered to IL-5-overexpressing mice that exhibit constitutive airway inflammation. Animals were subjected to whole-body plethysmography (WBP) and then killed for performance of bronchoalveolar lavage, histology, and serology. RESULTS: DEP delivery concomitant with OVA challenge or after the induction of airway inflammation with this allergen induced increased AHR in models 1 and 2, respectively. Although these animals showed DEP-induced inflammation and mucus production in the intermediary airways, there was no effect on OVA-specific IgE or T(H)2 cytokine production. In the IL-5 transgenic mice it was possible to induce similar effects with DEPs in the absence of an allergen. CONCLUSION: We demonstrate that DEPs induced AHR independent of their adjuvant effects, suggesting the use of these models to study the mechanism or mechanisms of acute asthma exacerbation by means of PM.     

Dectin-2 sensing of house dust mite is critical for the initiation of airway inflammation

How the immune system senses aeroallergens and triggers an aberrant inflammation is poorly understood. Dectin-2 is a house dust mite (HDM)-sensing pattern recognition receptor. In a 3-week mouse model of repeated intranasal HDM challenge, anti-Dectin-2 potently attenuated the characteristic allergic inflammation and airway hyper-responsiveness. Anti-Dectin-2 also prevented neutrophil influx following a single HDM challenge. Interestingly, cysteinyl leukotrienes, but not chemokine and cytokine levels were inhibited by anti-Dectin-2 in this acute model, and in ex vivo challenge of cultured alveolar macrophages with HDM. Furthermore in the single-challenge model, zileuton, an inhibitor of leukotriene production, produced a similar effect as Dectin-2 blockade. Together these data suggest alveolar macrophage sensing of HDM by Dectin-2 elicits the production of cysteinyl leukotrienes, and this axis is key for the initiation of airway inflammation to this aeroallergen. Finally, we found Dectin-2-positive infiltrating cells present in bronchial biopsies from asthmatic subjects.     

Effects of diesel exhaust particles and carbon black on induction of dust mite allergy in brown norway rats

Particulate matter (PM) components of air pollution have been associated with mortality and health risks in susceptible populations including asthmatics. More than a decade of PM research has demonstrated that these effects do not occur indiscriminately and are related to particle size, surface area, and chemical composition. Experimental evidence in rodents indicates that inhaled or instilled diesel exhaust particles (DEPs) increase lung injury, inflammation, and allergic airway responses, and that ultra-fine carbon black (UFCB) particles cause more pulmonary inflammation than fine carbon black (FCB) particles in a dose-dependent manner. Our preliminary work determined that a dose of 100 mug of FCB, UFCB, or DEPs (NIST SRM 2975) was sufficient to enhance pulmonary inflammation in Brown Norway (BN) rats 24 hours after intratracheal (IT) instillation of the particles. In the current investigation, we sought to compare, on a mass basis, the effects of a 100 mug dose of these particles on allergic sensitization to house dust mite (HDM) antigen. Immediate airway responses (IAR) to HDM challenge and a battery of proinflammatory, allergic, and acute injury responses in the lung were then measured two and seven days post-challenge. DEPs exposure increased 8 of 10 responses including IAR and levels of IL-4, IL-13, TNFalpha, total protein, cysteinyl leukotrienes, and eosinophils in bronchoalveolar lavage fluid (BALF). UFCB and FCB significantly enhanced 4 of 10 and 2 of 10 of these responses compared to saline, respectively. Among other responses that were not statistically elevated with particle treatment, mean values for FCB were higher than for UFCB. Particles administered prior to challenge rather than prior to sensitization did not significantly enhance any of these responses above levels of saline controls. We conclude that on a mass basis, DEPs had the greatest potential to enhance allergic induction, indicating that chemical composition is more important than particle size in determining potency for this health effect.     

Nonlinear relations between house dust mite allergen levels and mite sensitization in farm and nonfarm children

BACKGROUND: Low sensitization rates to common allergens have been observed in farm children, which might be due to high exposure to microbial agents. It is not known how microbial agents modify the association between specific allergen exposure and sensitization. OBJECTIVE: To examine the relations between house dust mite allergen exposure and mite sensitization in farm and nonfarm children and to assess the effects of microbial agents levels on this association. METHODS: Major mite allergens of Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1), endotoxin, beta(1,3)-glucans and fungal extracellular polysaccharides were measured in mattress dust of 402 children participating in a cross-sectional study in five European countries. Mite allergen (Der p 1 + Der f 1) levels were divided into tertiles with cut-offs 1.4 and 10.4 microg/g. Sensitization was assessed by measurement of allergen-specific immunoglobulin E against house dust mite. RESULTS: Prevalence ratios of mite sensitization for medium and high when compared with low mite allergen levels were 3.1 [1.7-5.7] and 1.4 [0.7-2.8] respectively. Highest mite sensitization rates at intermediate exposure levels were consistently observed across country (except for Sweden) and in both farm and nonfarm children. The shape of the dose-response curve was similar for above and below median mattress microbial agent levels, but the 'sensitization peak' appeared to be lower for above median levels. CONCLUSIONS: Our data suggest a bell-shaped dose-response relationship between mite allergen exposure and sensitization to mite allergens. In populations with high microbial agent levels and low sensitization rates, the curve is shifted down.     

慢性尘螨暴露诱导小鼠气道Th1炎症相关的气道重塑

目的:观察慢性尘螨变应原暴露对小鼠气道变应性炎症及重构的影响。方法:采用α平滑肌肌动蛋白启动子驱动的Cre重组酶(α-SMA-Cre)与R26R双转基因报告小鼠,尘螨连续滴鼻60 d后测定气道阻力;肺泡灌洗并分类计数;分离肺组织并提取肺组织蛋白;制备病理组织切片;分离培养脾脏淋巴细胞。结果:慢性尘螨暴露组小鼠气道阻力明显升高(P0.01),肺泡灌洗液(BALF)中细胞总数和淋巴细胞比例显著高于正常对照组(P0.01),肺组织HE染色示:慢性尘螨暴露组小鼠气道上皮细胞炎症水肿,小血管周围淋巴细胞浸润,未见嗜酸粒细胞。X-gal染色显示:尘螨暴露组气道平滑肌细胞及上皮下肌纤维母细胞明显增生,相应Western blotting结果显示α平滑肌肌动蛋白(α-SMA)表达明显增多。尘螨暴露组肺匀浆上清γ干扰素(IFN-γ)较正常对照组升高(P0.01),白细胞介素-4(IL-4)水平两者间没有显著差异(P0.05)。流式细胞检测显示尘螨暴露组分泌IFN-γ的CD4+Th1细胞较正常对照组增多。结论:慢性尘螨气道暴露诱导了Th1炎症,与气道重构和气道高反应性密切相关。     

Characterization of the house dust mite allergen Der p 7 by monoclonal antibodies

The house dust mite allergen Der p 7, which was defined by cDNA cloning, has been shown to react with about 50% of allergic sera and corresponds to or is antigenically related to at least three different sized components in mite extracts. To characterize these entities, monoclonal antibodies (MoAbs) were generated by immunizing BALB/c mice affinity-purified Der p 7-GST (glutathione S-transferase) fusion protein. MoAbs WH9 and WH22 showed positive reactivity to recombinant Der p 7 negative reactivity to GST and the Der p 5-GST fusion protein in ELISA and immunoblotting. The specificity of both MoAbs was confirmed by inhibition of the ELISA activity by recombinant Der p 7 but not by the recombinant Der p 5. Immunoblot analysis demonstrated that both MoAbs showed reactivities to components with molecular weights (mol. wt.) of 31, 30 and 26kDa reactive to both MoAbs. At least six major forms with different pI or size were indicated by 2-D gel analysis. In addition to characterization of Der p 7, both MoAbs may also be considered for use in the standardization of Der p 7 in mite extracts.     

Endotoxin and house dust mite allergen levels on synthetic and buckwheat pillows

Pillows are known to contain significant levels of indoor allergens and endotoxin, that are of importance to house dust mite sensitized asthmatics. Buckwheat pillows are commonly used in Korea. We studied the levels of the house dust mite allergen, Der f 1, and endotoxin on new synthetic and buckwheat pillows and their accumulation over three months. Endotoxin levels were significantly higher on new buckwheat pillows compared to synthetic pillows; geometric mean levels (95% CI) were 60,950 EU/g (30,270-122,700) and 4,887 EU/g (2,570-9,311) respectively (p<0.001). No Der f 1 was detected on the new pillows. After three months Der f 1 levels were similar on buckwheat and synthetic pillows, geometric mean levels (95% CI) were 1.16 microg/g (0.02-8.13) and 1.08 microg/g (0.19-1.68) respectively. These results indicate that buckwheat pillows are a source of very high endotoxin levels that may be of relevance to asthma severity of atopic asthmatics.